Veterans suffering loss of the larynx's vocal fold cover due to trauma or laryngeal cancer can suffer disabling voice difficulties, and treatment options are limited. A vibrating replacement tissue would revolutionize the treatment of laryngeal disorders. The work described in this proposal characterizes and optimizes a tissue-engineered vocal fold cover (TE-VFC). Adult human stem cells isolated from adipose tissue are cultured within fibrin hydrogel derived from the blood product cryoprecipitate. The result is a completely autologous three- dimensional tissue substitute. A significant innovation of this approach to vocal fold tissue engineering is th concomitant replication of the two layers (epithelial and mesenchymal) responsible for vibration in the native vocal fold cover. This model will be employed to systematically study fundamental issues of tissue engineering, stem cell differentiation, and vocal fold physiology. It is controllable, with an optimized case that most closely replicates normal developmental conditions and produces bilayered epithelial and mesenchymal differentiation. A control case is more typical of standard tissue culture conditions, and produces disorganized cell differentiation. Three research arms are proposed. First, extracellular matrix and basement membrane deposition are determined in the TE-VFC and the non- epithelialized control using immunohistochemistry and in situ hybridization. Epithelial barrier function is measured by trans-epithelial diffusion. Mechanisms for a difference in ECM remodeling between the two cases are investigated, such as matrix metalloproteinase secretion and pro-collagen mRNA. Second, mesenchymal cell phenotype is defined in the two conditions by protein expression and contractility. Potential signaling mechanisms controlling the phenotype are investigated, including EGF receptor and TGF- 1. Finally, vibration and phonation are assessed in both cases as well as in de-epithelialized TE-VFC to identify the role of epithelium in voice production. The constructs are attached to the vocal ligaments of excised larynges simulating implantation conditions, and airflow-induced vibration is recorded with high-speed imaging. Successful completion of the proposed research will determine whether including an epithelium in a developing tissue construct offers benefit, by comparing the cell phenotype, microstructure, and function in epithelialized and control cases. It will also identify mechanisms by which epithelial cells influence adjacent cell phenotype and ECM remodeling, in a controlled system. Findings will be relevant in other epithelialized tissues as well as this vocal fold replacement. Finally, these in vitro studies of the TE-VFC will provide necessary information to proceed with animal and human implantation trials to treat severe vocal fold scarring.